Biological reactions involve small numbers of molecules. Therefore all biological processes are prone to fluctuations, or 'noise," which creates cell-cell variation even within isogenic populations. It is not known whether noise in biological systems affects the fitness of an organism. The goals of this research proposal are to identify mechanisms that affect noise during MAP kinase signaling and to determine how stochastic effects on signal transduction influence the efficiency of biological responses. The specific aims are (1) to design and implement GFP-based reporters for signaling-based noise in the yeast pheromone-response MAPK pathway; (2) to screen the yeast deletion library and a library of mutants in the multi-kinase scaffold Ste5 for those that increase or decrease signaling-dependent noise; (3) to ascertain the biochemical function(s) of the genes identified; and (4) to determine the effects of increased or decreased noise on fitness using sensitive assays for mating efficiency. The identification of new population-level mechanisms of regulation of MAPK signaling in yeast may provide fundamental insight into the mechanisms and implications of intra-tumor heterogeneity, which currently poses a significant roadblock to effective cancer chemotherapy.